Low-dose ethanol consumption inhibits neutrophil extracellular traps formation to alleviate rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Ethanol consumption has been reported to reduce morbidity in RA patients, but the mechanism behind it remains unclear. Our results showed that Muribaculaceae was predominant in the gut microbiota of mice after ethanol treatment, and the levels of microbiota metabolite acetate were increased. Acetate reduced arthritis severity in collagen-induced arthritis (CIA) mice, which was associated with a decrease in the articular neutrophils and the myeloperoxidase-deoxyribonucleic acid complex in serum. Meanwhile, in vitro experiments confirmed that acetate affected neutrophil activity by acting on G-protein-coupled receptor 43, which reduced endoplasmic reticulum stress in neutrophils and inhibited neutrophil extracellular traps formation. Furthermore, exogenous acetate reversed CIA mice with exacerbated gut microbial disruption, further confirming that the effect of gut microbial metabolite acetate on neutrophils in vivo is crucial for the immune regulation. Our findings illuminate the metabolic and cellular mechanisms of the gut-joint axis in the regulation of autoimmune arthritis, and may offer alternative avenues to replicate or induce the joint-protective benefits of ethanol without associated detrimental effects.


Reporting on race, ethnicity, or other socially relevant groupings
No information regarding race,ethnicity,and other socially relevant groupings was collected from human participants.These actors are not related to our hypothesis,and only within-subject comparisons were made.

Population characteristics
No demographic information was collected from human participants.These factors are not related to our hypothesis,and only within-subject comparisons were made.

Recruitment
For the collection of human synovial tissue for analysis of joint pathology,written informed consent was obtained from patients recruited from department of orthopedics, the Second People's Hospital of Hefei.For the collection volunteers blood,flyers were posted in the hospital,and participants called researchers to volunteer.

Ethics oversight
All experimental procedure were conducted in accordance with ethical regulation and use in China.The experimental protocol involving human subjects was approved by the Clinical Medical Research Ethics Committee of the First Affiliated Hospital of Anhui Medical University (approval number: 2022275).
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
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Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample size is indicated in the figure legend for each experiments.For cell-based quantitative experiments, results of three independent biological replicates were used.For animal studies,we analyze a sufficient number of animals per group (minimum 5 animals) to evaluate differences between different groups.Statistical comparisons were performed using Student's t test for comparison between two groups or for paired comparisons, one-way ANOVA followed by Tukey post hoc test when more than two groups under same condition were involved, and two-way ANOVA followed by Sidak's or Tukey's post hoc test for comparison between two or more groups under two conditions.p-value less than 0.05 was considered significant.
Data exclusions No data were excluded from the experiments.

Replication
Experimental findings were replicated using biological replicates within and between independent experiments.In almost all cases,findings were validated in at least two independent experiments,with the exception being experiments that were deemed too time-or cost-intensive (e.g., metabolomics, survival)and could be supported and extended by complimentary techniques (e.g., in vitro surrogate assay).
Randomization Samples were randomly collected for each experimental group for analysis.Wherever possible, we collect all samples from each experimental condition and analyze them.

Blinding
Experimenters were blinded to score and grade of various indicators of mice; however, blinding was not always technically feasible.In these instances, samples were processed and/or positioned in an intercalated fashion to limit bias from group/batch effects.Subsequent data acquisition and analysis was performed in an automated fashion to reduce experimenter bias.
Reporting for specific materials, systems and methods DBA/1 mice were purchased from Gem Pharmatech (Nanjing, China).7-8-week-old male DBA/1 mice were used for experiments, which is the week age usually used in most RA modeling experiments.All mice were maintained under specific pathogen-free conditions at 25 with 12 h light and dark cycles in accordance with current ethical regulations for animal care and use in China.In the experiment, caging bias was taken into account.Instead of feeding them in an independent ventilated cage, they were all placed in the same environment open to the outside.

Wild animals
This study did not involve wild animals.

Reporting on sex
It is reported that no gender difference in CIA mice (Brand et al., 2007).In order to avoid the impact of various hormone fluctuations, such as estrogen, in the female menstrual cycle on the pathogenesis of RA, only male mice are used as experimental subjects (Miyoshi et al.,2018).
Field-collected samples The study did not involve samples collected from the field.

Ethics oversight
This study was approved by the Experimental Animal Ethics Committee of Anhui Medical University (approval number: 20220454).
Animal welfare and experimental procedures were strictly in accordance with the guidelines for the care and use of laboratory animals.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Novel plant genotypes
Describe the methods by which all novel plant genotypes were produced.This includes those generated by transgenic approaches, gene editing, chemical/radiation-based mutagenesis and hybridization.For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed.For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor was applied.

Seed stocks
Report on the source of all seed stocks or other plant material used.If applicable, state the seed stock centre and catalogue number.If plant specimens were collected from the field, describe the collection location, date and sampling procedures.

Authentication
Describe any authentication procedures for each seed stock used or novel genotype generated.Describe any experiments used to assess the effect of a mutation and, where applicable, how potential secondary effects (e.g.second site T-DNA insertions, mosiacism, off-target gene editing) were examined.

Plants Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
The mouse feet were cut into tissue blocks of 3-4 mm in a 1.5 mL EP tube, and added Hank's equilibrium salt solution (1 mL), collagenase type II (1 g/mL, 2 μL) and CaCl2 solution (3 mM, 5 μL).After incubated at 37 for 4 h, taken the supernatant by centrifugation, washed with PBS 3 times after passing through a nylon sieve to obtain dispersed cells.Fluorescent antibodies were added to the treated single cell suspension (1×10^6/100 μL), and incubated at 4°C in the dark for 30 min.Then, after centrifugation at 300 g, PBS was added and the cells were repeatedly washed 3 times.Finally, 300 μL PBS was added to resuspend, and then the results of cell sorting were observed.

Instrument
Flow cytometry acquisition was performed using an BD FACSCanto VerseII (BD Bioscience).

Cell population abundance
Approximately 100,000-300,000 events of interest were captured per sample.For MACS sorted cells, the purity of relevant population was validated by FACS analysis.

Gating strategy
Cells were gated for lymphocyte population (FSC-A/SSC-A), then singlets were gated in FSC-W/FSC-H and SSC-W/SSC-H and live cells were selected by Live/Dead Fixable Dye.We designed the experiment with reference to the work of Shin AE.
et al. (Gastroenterology.2023) and Huang et al.(Hepatology.2022), B cells (CD45+CD19+), mature T lymphocytes (CD3+CD4+), Animals and other research organismsPolicy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research